With increased development activities in recent years in nucleic acid pharmaceutical, there is a need for a technology for the analysis of oligonucleotide sequences of about several dozen bases long. With nucleic acid drugs, a general practice is to increase in vivo retention by the introduction of an artificial moiety such as phosphorothioate esters and modified ribose 2′-OH groups. There is a need for a technology that can sequence nucleic acids that include an artificial moiety.
Examples of the use of MALDI-TOF MS to sequence oligonucleotides include a method (see Non-Patent Literature 1) wherein a ladder structure mass spectrum is obtained for RNA whose phosphodiesters have been partially hydrolyzed with an acid and the sequence is analyzed based on the mass difference between peaks, and a method (see Non-Patent Literature 2) wherein a RNA is sequentially decomposed with an endonuclease starting from either the 3′-end or the 5′-end, mass spectra are obtained over time and sequence information is obtained.
In-source decay (ISD) and MALDI-TOF MS are primarily used for the analysis of amino acid sequence of peptides (see Non-Patent Literature 3 and 4). However, there are several reported cases of their use for the analysis of nucleic acid base sequence (see Non-Patent Literature 5 and 6).
With respect to DNA that is 11 bases long, Non-Patent Literature 5 discloses the generation of fragment ions by irradiation with laser of a wavelength of 266 nm while using picolinic acid as a matrix.
With respect to DNA that is 7 bases long, Non-Patent Literature 6 discloses the generation of fragment ions using 2,5-dihydroxybenzoic acid (2,5-DI-113) as a matrix. Even though the literature does not identify the laser wavelength, based on the use of Voyager Elite (manufactured by Perspective Biosystems), the wavelength is estimated to be 337 nm.
Non-Patent Literature 7 discloses the analysis of a DNA sequence by in-source decay using a mixture of dihydroxyacetophenone (DHAP) and 1,5-diaminonaphtalene (DAN) as a matrix. The literature states that almost no fragments were detected when in-source decay was used on RNA.
Non-Patent Literature 8 discloses the use of 2,4-dihydroxyacetophenonematrix for the separation of 100-base long DNA and 102-base long DNA consisting of TC repeat sequences on a mass spectrum and their detection.
With base sequence analysis of nucleic acids using mass spectrometry, reflecting the cleavage site of the phosphodiester bonds, fragment ions that are generated from the nucleic acid are named as a, b, c, or d if they possess a 5′-OH group and as w, x, y or z if they possess a 3′-OH group (see Non-Patent Literature 9).
However, there are no reports of the analysis of sequences of RNAs (unmodified) and RNAs having modified groups based on the mass spectrum of fragment ions that are generated by in-source decay.